Roseola infantum (exanthem subitum) is a childhood illness which manifests itself in a short period of high fever followed by a mild, disseminated rash. After approximately four days, symptoms are gone. Rarely does roseola itself present complications. The etiologic agent of roseola is human herpesvirus 6 (HHV-6). Like other herpesviruses, HHV-6 appears to lie latent in the host. The site and effects of latent infection and symptoms or disease associated with reactivation are unknown. To develop tools to address these and other questions of the biology of HHV-6 as well as to begin to understand how the molecular details of HHV-6 compare with those of other herpesviruses, this project will continue structural analysis of the HHV-6 genome and determine the feasibility of a functional analysis of the HHV-6 genomic termini. Channel catfish virus (CCV) is a herpesvirus which is unrelated to HHV-6 with respect to its normal host and its subfamily classification. However, CCV exhibits the same general genomic architecture as HHV-6. By comparing the repeated DNA sequence elements found at the genomic termini of these diverse herpesviruses, it is hoped that some insight will be gained into their functions. Terminal restriction fragments from HHV-6 and CCV will be cloned into bacterial plasmids using methods designed to maintain the terminal structures. At least two methods will be employed, one utilized by others to clone the termini of varicella-zoster virus and another, novel method based on synthetic oligonucleotide linkers. In 1984, A.J. Davison reported the cloning and sequencing of varicella-zoster virus terminal fragments using a homopolymer tailing method. Separate clones were generated after tailing genomic termini with deoxycytidine or deoxyguanine. By comparing the clones from each method, single base 3' extensions were identified. Alternatively, oligonucleotide linkers will be generated containing single base 3' extensions predicted to be complementary to the genomic termini. After ligation to viral DNA, the linkers will be cleaved at a restriction site within their sequence to liberate a fragment with ends homologous to the restricted ends of the plasmid vector. DNA sequences will be determined by dideoxynucleotide sequencing and analyzed for potential protein coding sequences and homologies between the two viruses. Finally, the feasibility of initiating functional analysis by generating HHV-6/CCV amplicons and intertypic recombinants will be explored.